21st biotechnology presents the possibility of new solutions to the food, environment and health problems, with the ultimate object of promoting human prosperity. In recent years, the technology of using stem cells has been considered as a new way to treat incurable diseases. Formerly, organ transplantation, gene therapy, etc., were presented for the treatment of incurable human diseases, but their efficient use has not been made due to immune rejection, a short supply of organs, an insufficient development of vectors, and an insufficient knowledge of disease genes.
For this reason, with increasing interests in stem cell studies, it has been recognized that totipotent stem cells having the ability to form all the organs by proliferation and differentiation can not only treat most of diseases but also fundamentally heal organ injuries. Also, many scientists have suggested the applicability of stem cells for the regeneration of all the organs and the treatment of incurable diseases, including Parkinson's disease, various cancers, diabetes and spinal damages.
Stem cells refers to cells having not only self-replication ability but also the ability to differentiate into at least two cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
Totipotent stem cells are cells having totipotent properties capable of developing into one perfect individual, and these properties are possessed by cells up to the 8-cell stage after the fertilization of an oocyte and a sperm. When these cells are isolated and transplanted into the uterus, they can develop into one perfect individual.
Pluripotent stem cells, which are cells capable of developing into various cells and tissues derived from the ectodermal, mesodermal and endodermal layers, are derived from an inner cell mass located inside of blastocysts generated 4-5 days after fertilization. These cells are called “embryonic stem cells” and can differentiate into various other tissue cells but not form new living organisms.
Multipotent stem cells, which are stem cells capable of differentiating into only cells specific to tissues and organs containing these cells, are involved not only in the growth and development of various tissues and organs in the fetal, neonatal and adult periods but also in the maintenance of homeostasis of adult tissue and the function of inducing regeneration upon tissue damage. Tissue-specific multipotent cells are collectively called “adult stem cells”.
Adult stem cells are obtained by taking cells from various human organs and developing the cells into stem cells and are characterized in that they differentiate into only specific tissues. However, recently, experiments for differentiating adult stem cells into various tissues, including liver cells, were dramatically successful.
The multipotent stem cells were first isolated from adult marrow (Jiang et al., Nature, 418:41, 2002), and then also found in other various adult tissues (Verfaillie, Trends Cell Biol., 12:502, 2002). In other words, although the marrow is the most widely known source of stem cells, the multipotent stem cells were also found in the skin, blood vessels, muscles and brains (Tomas et al., Nat. Cell Biol., 3:778, 2001; Sampaolesi et al., Science, 301:487, 2003; Jiang et al., Exp. Hematol., 30:896, 2002). However, stem cells in adult tissues, such as the marrow, are very rarely present, and such cells are difficult to culture without inducing differentiation, and so difficult to culture in the absence of specifically screened media. Namely, it is very difficult to maintain the isolated stem cells in vitro.
Recently, adipose tissue was found to be a new source of multipotent stem cells (Cousin et al., BBRC., 301:1016, 2003; Miranville et al., Circulation, 110:349, 2004; Gronthos et al., J. Cell Physiol., 189:54, 2001; Seo et al., BBRC., 328:258, 2005). Namely, it was reported that a group of undifferentiated cells is included in human adipose tissue obtained by liposuction and has the ability to differentiate into fat cells, osteogenic cells, myoblasts and chondroblasts (Zuk et al., Tissue Eng., 7:211, 2001; Rodriguez et al., BBRC., 315:255, 2004). This adipose tissue has an advantage in that it can be extracted in large amounts, and thus, it receives attention as a new source of stem cells, which overcomes the existing shortcomings.
Also, recent studies using animal model experiments indicate that adipose tissue-derived cells have the abilities to regenerate muscles and to stimulate the differentiation of nerve blood vessels. Thus, these adipose tissue-derived cells have attention as a new source of stem cells.
Adipose tissue-derived stem cells known till now include human adipose-derived adult stem cells that can differentiate into epithelial cells (Brzoska et al., BBRC, 330:142, 2005), human adipose-derived adult stem cells that can differentiate into osteogenic and fat cells (Cao et al., BBRC, 332:370, 2005), human adipose-derived adult stem cells that can differentiate into nerve cells (Safford et al., BBRC, 294:371, 2002), rat adipose-derived stem cells that can differentiate into fat cells (Ogawa et al., BBRC, 319:511, 2004), rat adipose-derived stem cells that can differentiate into osteogenic and chondrogenic cells (Ogawa et al., BBRC, 313:871, 2004), human adipose-derived stem cells that can differentiate into cartilage cells (Biomaterials, 25:3211, 2004), rat adipose-derived stem cells that can differentiate into nerve cells (Fujimura et al., BBRC, 333:116, 2005), and adipose-derived stem cells that can differentiate into bone cells, cartilage cells, nerve cells or muscle cells (U.S. Pat. No. 6,777,231).
However, most of adipose-derived stem cells known till now are stem cells derived from the adipose tissue of animals other than human beings. Even if they are stem cells derived from human adipose tissue, they have been limited to those derived from tissues obtained by the liposuction of abdominal fat, and the kind of cells differentiated from the stem cells has also been limited. Particularly, isolated stem cells have low proliferation rates and are difficult to maintain in an undifferentiated state for a long period of time, and thus, have been limited in application.
Accordingly, the present inventors have made extensive efforts to develop multipotent adult stem cells, which have high proliferation rates, can be maintained in a undifferentiated state for a long period of time by forming spheres and can differentiate into more various cells, as a result, found that multipotent stem cells isolated from human adipose tissue can differentiate into various cells, including osteogenic cells, chondrogenic cells, nerve cells, astrocytes, fat cells, and insulin-releasing pancreatic beta-cells, have a very high proliferation rate and can be maintained in an undifferentiated state for a long period of time by forming spheres, thereby completing the present invention.